SUIT-020: Difference between revisions
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== Specific SUIT protocols == | == Specific SUIT protocols == | ||
[[File:1PM;2D;3G;4S;5Rot;6Omy;7U;8Ama.png|300px]] | [[File:1PM;2D;3G;4S;5Rot;6Omy;7U;8Ama.png|300px]] | ||
[[File:SUIT-020 | [[File:SUIT-020 Fluo mt D033 O2 DL-File2.png|600px]] | ||
[[File:SUIT-020 Fluo mt D033 DL- | [[File:SUIT-020 Fluo mt D033 DL-File2.png|600px]] | ||
:::* [[SUIT-020 Fluo mt D033]]ย simultaneous determination of O<sub>2</sub> flux and mt-membrane potential using any fluorescence dye ([[Safranin]], [[TMRM]] etc) on isolated mitochondria and tissue homogenate.ย ย | :::* [[SUIT-020 Fluo mt D033]]ย simultaneous determination of O<sub>2</sub> flux and mt-membrane potential using any fluorescence dye ([[Safranin]], [[TMRM]] etc) on isolated mitochondria and tissue homogenate.ย ย | ||
[[File:1GM;2D;3S;4Rot;5Omy;6U;7Ama.png|300px]] | [[File:1GM;2D;3S;4Rot;5Omy;6U;7Ama.png|300px]] |
Revision as of 12:21, 28 February 2019
Description
File:SUIT-020 O2 mt D032.png File:SUIT-020 O2 mt D035.png
Abbreviation: NS
Reference: A: simultaneous determination of O2 flux and mt-membrane potential ยปVersions
- SUIT-category: NS
- SUIT protocol pattern: orthogonal 1PM;2D;3G;4S;5Rot;6Omy;7U;- and 1GM;2D;3S;4Rot;5Omy;6U;-
This is a protocol to investigate the N- and NS-pathway control state on isolated mitochondria and tissue homogenate. The protocol can be applied to cells or tissue types that display a preference for either GM over PM to support NADH-linked respiration or vice versa. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be determined. This is an ideal protocol for simultaneous determination of O2 flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. SUIT-020 O2 mt D032 and SUIT-020 O2 mt D035 are protocols which can be performed without the fluorescence dye as well.
Communicated by Huete-Ortega M, Komlodi T and Gnaiger E (last update 2019-02-20)
Specific SUIT protocols
- SUIT-020 Fluo mt D033 simultaneous determination of O2 flux and mt-membrane potential using any fluorescence dye (Safranin, TMRM etc) on isolated mitochondria and tissue homogenate.
- SUIT-020 Fluo mt D036 simultaneous determination of O2 flux and mt-membrane potential using any fluorescence dye (Safranin, TMRM, etc) on isolated mitochondria and tissue homogenate.
- SUIT-020 O2 mt D032 determination of O2 flux on isolated mitochondria and tissue homogenate as a control for SUIT-020 Fluo mt D033.
- SUIT-020 O2 mt D035 determination of O2 flux on isolated mitochondria and tissue homogenate as a control for SUIT-020 Fluo mt D036.
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1PM | PML(n) | N | CI | 1PM
|
2D | PMP | N | CI | 1PM;2D
|
2c | PMcP | N | CI | 1PM;2D;2c
|
3G | PGMP | N | CI | 1PM;2D;2c;3G
|
4S | PGMSP | NS | CI&II | 1PM;2D;2c;3G;4S
|
5Rot | SP | S | CII | 1PM;2D;2c;3G;4S;5Rot
|
6Omy | SL(Omy) | 1PM;2D;2c;3G;4S;5Rot;6Omy
| ||
7U* | SE | S | CII | 1PM;2D;2c;3G;4S;5Rot;6Omy;7S
|
8Ama | ROX | 1PM;2D;2c;3G;4S;5Rot;6Omy;7S;8Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1GM | GML(n) | N | CI | 1GM
|
2D | GMP | N | CI | 1GM;2D
|
2c | GMcP | N | CI | 1GM;2D;2c
|
3S | GMSP | NS | CI&II | 1GM;2D;2c;3S
|
4Rot | SP | S | CII | 1GM;2D;2c;3S;4Rot
|
5Omy | SL(Omy) | 1GM;2D;2c;3S;4Rot;5Omy
| ||
6U* | SE | S | CII | 1GM;2D;2c;3S;4Rot;5Omy;6U
|
7Ama | ROX | 1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- + You can get information with a single protocol about the NS, S and N-pathway control state.
- + The protocol can be combined with fluorescence dyes to evaluate mitochondrial membrane potential.
- - Many fluorescence dyes (such as Safranin, TMRM etc) can inhibit components of the ET system, most commonly affecting NADH-linked respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. The following protocols can be used: SUIT-020 O2 mt D032, SUIT-020 O2 mt D035.
- - CIV activity cannot be determined and Cytochrome c test cannot be performed together with the fluorescence dyes.
Compare SUIT protocols
- SUIT-001;SUIT-001 O2 mt D001for isolated mitochondria and tissue homogenate: a complex protocol to get information not only about the NS(PGM) in the OXPHOS state, but also in ET state and about the FGp-pathways additionally.
- SUIT-004;SUIT-004 O2 pfi D010: it provides an information about the NS(PM) pathway in the ET state withou the contribution of G.
- SUIT-008;SUIT-008 O2 pfi D014 and SUIT-008 O2 pce D025: it provides information about the NS(PGM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
- SUIT-011:it provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
- SUIT-012:it provides information about the N(PGM) pathway in the OXPHOS and ET-states wihtout the contribution of S-pathway and without the addtion of Omy.
- SUIT-014: it is similar protocol to SUIT-020 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet20.13 Safranin mt-membranepotential | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with safranin or TMRM. | Mouse | Nervous system |
Krumschnabel 2014 Methods Enzymol | 2014 | Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1 | Mouse | Nervous system |
MitoPedia concepts: MiP concept, SUIT protocol, Recommended
MitoPedia methods:
Respirometry,
Fluorometry